Bottom Line:
At least a 30-min heat shock is required for the expression of a protein of interest and a 60-min heat shock yields the maximum level of protein expression.After the heat shock, the mRNA level decreases rapidly.Expression of the dominant negative DRP5B resulted in the appearance of aberrant-shaped cells in which two daughter chloroplasts and the cells are still connected by a small DRP5B positive tube-like structure.

ABSTRACTThe cell of the unicellular red alga Cyanidioschyzon merolae contains a single chloroplast and mitochondrion, the division of which is tightly synchronized by a light/dark cycle. The genome content is extremely simple, with a low level of genetic redundancy, in photosynthetic eukaryotes. In addition, transient transformation and stable transformation by homologous recombination have been reported. However, for molecular genetic analyses of phenomena that are essential for cellular growth and survival, inducible gene expression/suppression systems are needed. Here, we report the development of a heat-shock inducible gene expression system in C. merolae. CMJ101C, encoding a small heat shock protein, is transcribed only when cells are exposed to an elevated temperature. Using a superfolder GFP as a reporter protein, the 200-bp upstream region of CMJ101C orf was determined to be the optimal promoter for heat-shock induction. The optimal temperature to induce expression is 50°C, at which C. merolae cells are able to proliferate. At least a 30-min heat shock is required for the expression of a protein of interest and a 60-min heat shock yields the maximum level of protein expression. After the heat shock, the mRNA level decreases rapidly. As an example of the system, the expression of a dominant negative form of chloroplast division DRP5B protein, which has a mutation in the GTPase domain, was induced. Expression of the dominant negative DRP5B resulted in the appearance of aberrant-shaped cells in which two daughter chloroplasts and the cells are still connected by a small DRP5B positive tube-like structure. This result suggests that the dominant negative DRP5B inhibited the final scission of the chloroplast division site, but not the earlier stages of division site constriction. It is also suggested that cell cycle progression is not arrested by the impairment of chloroplast division at the final stage.

pone-0111261-g004: Basal transcriptional activities of the upstream region of CMJ101C orf at lower temperatures in the stable transformants.A single copy of sfGFP orf fused with the 200 (S-200), 250 (S-250) or 345 (S-345)-bp upstream region of CMJ101 orf (Figure 3A) was integrated into the C. merolae genomic-neutral locus [11]. The stable transformants were cultured at 35∼45°C for 1 day. (A) Semi-quantitative RT-PCR showing the level of the sfGFP mRNA. CMJ101C and TIM13 (CMB148C) were used as a positive and quantitative control, respectively. (B) Micrographs showing the GFP fluorescence and autofluorescence of chlorophyll (red). The scale bar is 10 µm.

Mentions:
To evaluate the basal level of GFP expression before the induction by heat shock, the S-200, S-250 and S-345 stable transformants were cultured at 35, 37, 40, 42, and 45°C for one day. It was shown by semi-quantitative RT-PCR that GFP mRNA was detected in the S-200 cells at only 42°C and 45°C. In contrast, in the S-250 and S-345 cells, the mRNA was detected at all of the temperatures tested, even though a higher level of mRNA was detected at 42°C and 45°C (Figure 4A). Consistent with the results of RT-PCR, GFP fluorescence was detected in the S-200 cells at only 42°C and 45°C. In contrast, some of the S-250 and S-345 cells exhibited GFP fluorescence at lower temperatures (S-250 at 35, 37 and 40°C, and S-345 at 37 and 40°C)(Figure 4B). The strongest fluorescence was observed at 45°C in all of the transformants. These results indicate that the 200-bp upstream region of CMJ101C orf is the most suitable for the heat shock inducible system in the sense of there being little leakage of expression at lower temperatures.

pone-0111261-g004: Basal transcriptional activities of the upstream region of CMJ101C orf at lower temperatures in the stable transformants.A single copy of sfGFP orf fused with the 200 (S-200), 250 (S-250) or 345 (S-345)-bp upstream region of CMJ101 orf (Figure 3A) was integrated into the C. merolae genomic-neutral locus [11]. The stable transformants were cultured at 35∼45°C for 1 day. (A) Semi-quantitative RT-PCR showing the level of the sfGFP mRNA. CMJ101C and TIM13 (CMB148C) were used as a positive and quantitative control, respectively. (B) Micrographs showing the GFP fluorescence and autofluorescence of chlorophyll (red). The scale bar is 10 µm.

Mentions:
To evaluate the basal level of GFP expression before the induction by heat shock, the S-200, S-250 and S-345 stable transformants were cultured at 35, 37, 40, 42, and 45°C for one day. It was shown by semi-quantitative RT-PCR that GFP mRNA was detected in the S-200 cells at only 42°C and 45°C. In contrast, in the S-250 and S-345 cells, the mRNA was detected at all of the temperatures tested, even though a higher level of mRNA was detected at 42°C and 45°C (Figure 4A). Consistent with the results of RT-PCR, GFP fluorescence was detected in the S-200 cells at only 42°C and 45°C. In contrast, some of the S-250 and S-345 cells exhibited GFP fluorescence at lower temperatures (S-250 at 35, 37 and 40°C, and S-345 at 37 and 40°C)(Figure 4B). The strongest fluorescence was observed at 45°C in all of the transformants. These results indicate that the 200-bp upstream region of CMJ101C orf is the most suitable for the heat shock inducible system in the sense of there being little leakage of expression at lower temperatures.

Bottom Line:
At least a 30-min heat shock is required for the expression of a protein of interest and a 60-min heat shock yields the maximum level of protein expression.After the heat shock, the mRNA level decreases rapidly.Expression of the dominant negative DRP5B resulted in the appearance of aberrant-shaped cells in which two daughter chloroplasts and the cells are still connected by a small DRP5B positive tube-like structure.

ABSTRACTThe cell of the unicellular red alga Cyanidioschyzon merolae contains a single chloroplast and mitochondrion, the division of which is tightly synchronized by a light/dark cycle. The genome content is extremely simple, with a low level of genetic redundancy, in photosynthetic eukaryotes. In addition, transient transformation and stable transformation by homologous recombination have been reported. However, for molecular genetic analyses of phenomena that are essential for cellular growth and survival, inducible gene expression/suppression systems are needed. Here, we report the development of a heat-shock inducible gene expression system in C. merolae. CMJ101C, encoding a small heat shock protein, is transcribed only when cells are exposed to an elevated temperature. Using a superfolder GFP as a reporter protein, the 200-bp upstream region of CMJ101C orf was determined to be the optimal promoter for heat-shock induction. The optimal temperature to induce expression is 50°C, at which C. merolae cells are able to proliferate. At least a 30-min heat shock is required for the expression of a protein of interest and a 60-min heat shock yields the maximum level of protein expression. After the heat shock, the mRNA level decreases rapidly. As an example of the system, the expression of a dominant negative form of chloroplast division DRP5B protein, which has a mutation in the GTPase domain, was induced. Expression of the dominant negative DRP5B resulted in the appearance of aberrant-shaped cells in which two daughter chloroplasts and the cells are still connected by a small DRP5B positive tube-like structure. This result suggests that the dominant negative DRP5B inhibited the final scission of the chloroplast division site, but not the earlier stages of division site constriction. It is also suggested that cell cycle progression is not arrested by the impairment of chloroplast division at the final stage.